**5. Conclusions**

*Leishmania (V.) braziliensis* is amenable to reverse genetics using a CRISPR–Cas9 protocol as shown in this work. Gene replacement occurs exclusively at the predicted sites. As is known, ectopic expression of the genes of interest presents a problem, due to the effects of RNAi in the *Viannia* subgenus. The functions of at least two amastigote-specific heat shock proteins, HSP100 and HSP23, are conserved between Old World and New World leishmaniae and likely in *T. brucei* as well. With a workable protocol for gene replacement now in place, urgent questions pertaining to the biology of the *Viannia* subgenus can now be addressed by means of reverse genetics.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/10/1159/s1, Figure S1: Detection of Cas9 and T7 RNAP in the *L. braziliensis* parental cell line; Figure S2: Flow cytometric analysis results for *L. donovani* eGFP-Cas9-expressing promastigotes transfected with *eGFP*-targeting sgRNAs; Figure S3: Flow cytometric analysis results for *L. braziliensis* eGFP-Cas9-expressing promastigotes transfected with *eGFP*-targeting sgRNAs; Figure S4: PCR verification of *eGFP* gene replacement; Figure S5: Titration of selection antibiotics on *L. braziliensis* promastigotes; Figure S6: Verification of the *L. braziliensis HSP23* gene replacement by the respective resistance cassettes; Figure S7: Verification of the *L. braziliensis HSP100* gene replacement by the respective resistance cassettes; Figure S8: Cas9 expression in *L. braziliensis HSP23*- and *HSP100*-null mutants; Figure S9: Karyotype analysis of *L. braziliensis* and *L. major* strains; Figure S10: Verification of *L. major HSP23*-null mutants; Figure S11: Verification of replacement cassette integration into the *L.major HSP23* locus; Figure S12: Verification of *L.major HSP23*-/ complementation lines; Table S1: List of primers used in this study; Table S2: Sequence identity analysis of trypanosomatid HSP23 proteins.

**Author Contributions:** V.A.: Study conception: generation and phenotype analysis of *L. braziliensis* mutants, manuscript preparation; C.K.-B.: Study conception, generation and phenotype analysis of *L. major* mutants, manuscript preparation; C.B.: Gene replacements: phenotype analysis, in vitro infections, next generation sequencing; H.Z.: Experimental design, data analysis; J.S.: FACS analysis; J.A.: Study conception: manuscript preparation; J.-C.D.: Study conception, manuscript preparation; J.C.: Study conception: experimental design, supervision, manuscript preparation. All authors have read and agreed to the published version of the manuscript.

**Funding:** V.A. was supported by a Humboldt Research Fellowship for Postdoctoral Researchers from the Alexander von Humboldt Foundation, Germany, during the study period. The publication of this article was funded by the Open Access Fund of the Leibniz Association.

**Acknowledgments:** We thank Andrea MacDonald and Dorothea Zander–Dinse (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) for technical assistance; Hideo Imamura (Institute of Tropical Medicine, Antwerp, Belgium), for providing the genome sequence of the *L. braziliensis* strain PER005cl2 and the novel long–read assembly of the *L. braziliensis* M2904 reference genome; Eva Gluenz and Tom Beneke (University of Oxford, UK), for providing the CRISPR–Cas9 toolkit plasmids developed for *Leishmania* spp.

**Conflicts of Interest:** The authors are not aware of any conflict of interest.
